Megalin and the low-density lipoprotein (LDL) receptor-related protein (LRP) are two large membrane glycoproteins that belong to the LDL receptor gene family. In addition to lipoprotein particles, LRP and megalin also bind and endocytose a variety of structurally and functionally distinct ligands. In polarized cells such as neurons, LRP is localized in the basal lateral/somatodendritic domain, whereas megalin is more restricted to the apical domain of the plasma membrane. Recently, new information has emerged regarding the structural and functional elements within the cytoplasmic tails of the LDL-R family members. Studies from our laboratories have shown that the YATL sequence and the distal di-leucine motif function as the endocytosis signals for LRP. These and other sequence motifs within the LRP tail are similar to the sorting signals present in basolateral proteins expressed in epithelial cells or somatodendritic proteins in neurons. Megalin's cytoplasmic tail is about twice the size of that of LRP, and contains several putative tyrosine-based or di-leucine endocytosis motifs, a proline-rich sequence with homology to a SH3 binding domain, a SEV motif that mediates binding to PDZ proteins, and several putative protein kinase consensus motifs. Because of its role in capturing ligands at the lumenal domain of several epithelia, in addition to internalizing and degrading its ligands, megalin can also transport some of them to the basolateral pole via a transcytosis process. One unexplored aspect in the study of these receptors is the molecular and cellular determinants that dictate their polarized distribution and intracellular trafficking in polarized cells. We hypothesize that LRP is expressed primarily on the basolateral surface of epithelial cells, and its basolateral sorting motif(s) are present in its cytosolic tail. The basolateral expression of LRP is the result of both sorting at the TGN and the early/recycling endosome. Megalin, on the other hand, is expressed predominantly on the apical side of epithelial cells and has different ways to achieve its polarized localization, including targeting, apical retention through tail interactions with the actin cytoskeleton, and association to lipid rafts. The specific aims of the current proposal are 1) to identify and characterize the sorting motif(s) within the cytoplasmic tail of LRP and within the cytoplasmic and/or transmembrane domain of megalin; 2) to study the cellular machinery involved in LRP and megalin sorting; and 3) to analyze the molecular determinants of the endocytic and transcytotic pathways of megalin in polarized epithelial cells. The current proposed research will be performed primarily in Chile as an extension of NIH grant DK61761 (Guojun Bu, PI). Results from these collaborative studies should allow us to elucidate molecular and cellular determinants dictating the differential sorting of megalin and LRP in polarized cells.